It is well documented in clinical reports that some individuals are more vulnerable than others to developing chronic pain with considerable variability in response to treatment with pain drugs. However, this individual pain vulnerability and differential drug response are rarely addressed in preclinical research on animal pain models. The individual pain vulnerability is thought to be largely mediated by epigenetic changes that are induced by prior stressful events and pain experience, and lead to differential responses to subsequent pain stimuli and analgesic drugs. This proposal is designed to investigate the role of DNA methylation, a long- lasting form of chromatin modification that mediates gene repression, in individual variance and vulnerability to developing chronic pain. In preliminary studies, we found that animals display considerable variance in developing negative emotions of affective pain under chronic pain and stress conditions; and the vulnerability to developing the negative emotions is closely associated with downregulation of DNA methyltransferases 3a (Dnmt3a) that catalyzes de novo DNA methylation in pain- and emotion-regulating central amygdala (CeA). We propose multidisciplinary studies to test our central hypothesis that pain- and stress-induced Dnmt3a downregulation and associated DNA hypomethylation in central amygdala is a key process that leads to increased individual vulnerability to developing chronic pain. Aim 1 will systematically characterize the individual variance and vulnerability in rat and mouse models of orofacial pain and establish a functional link between pain and stress by determining whether animals vulnerable to chronic pain are also vulnerable to stress in developing the negative emotions of affective pain. Aim 2 will determine the functional role of CeA Dnmt3a in the individual vulnerability by associating the downregulation of CeA Dnmt3a with the individual vulnerability and by mimicking the effect of pain and stress with functional knockdown of CeA Dnmt3a. Aim 3 will determine molecular manipulations that reduce the individual vulnerability by reversing pain-induced downregulation of CeA Dnmt3a and examine the function of pain inputs onto CeA neurons in induction of the pain vulnerability. These proposed studies are expected to provide key knowledge on the role of DNA methylation in the individual vulnerability and to move the field forward with potential molecular targets for the future development of individually targeted pain medicine.